Acute myeloid leukemia (AML) is primarily a disease of older adults with poor treatment outcomes. Despite years of intensive research, the standard induction therapy for AML has remained largely unchanged for decades. Thus, the development of new and efficacious therapeutic targets for AML is urgently needed. Leukemia cells exhibit multiple metabolic aberrations that may be therapeutically targeted. Here, we show that nicotinamide adenine dinucleotide (NAD+) promotes leukemogenesis and causes chemotherapy treatment resistance through fueling energetic metabolism, and pinpoints nicotinamide nucleotide adenylyltransferase 1 (NMNAT1) is a novel therapeutic target for AML.
To identify novel genes essential for AML, we performed a whole genome CRISPR dropout screen by using MOLM13 cell line and identified 1,951 essential genes (Fig. A). By searching druggable targets among these genes, we narrowed down to 345 genes, among which we found two genes, NMNAT1 (nicotinamide nucleotide adenylyltransferase 1) and NAMPT (nicotinamide phosphoribosyltransferase), both involved in key steps in NAD+ biosynthesis. We comprehensively analyzed dependency scores for all genes involved in the NAD+ biosynthetic pathways (de novo synthesis pathway, the Preiss-Handler pathway and the salvage pathway) across a broad panel of cancer cell lines from the Dependency Map database (https://depmap.org/portal/). The results showed that NMNAT1 and NAMPT are both strongly selective and uniquely required for hematological malignancies compared to other cancers (Fig. B). Since little success has been achieved for NAMPT inhibitors in clinical trials, our attention was drawn to NMNAT1, which encodes a nuclear localized enzyme catalyzing the final step in NAD+ biosynthesis. We confirmed that deletion of NMNAT1 in AML cells significantly reduced nuclear NAD+ level and cell viability over time while sparing normal hematopoietic progenitor cells, suggesting that NMNAT1 is targetable to AML. Overexpression of wild-type Nmnat1 but not the enzymatically inactive forms rescued NMNAT1-KO AML, indicating that the catalytic activity of NMNAT1 is required for AML.
To study the role of NAD+ in AML, we first measured NAD+ levels in leukemic and normal cells, and found higher NAD+ levels in leukemia-initiating cells from a murine MLL-AF9-induced AML model compared to normal cells. Supplementation of NAD+ metabolites (NMN, NAM and NR) increased AML proliferation, enhanced glycolysis (lactate production) and oxidative phosphorylation (ATP production), resulting in chemotherapy resistance (Fig. C). Deletion of NMNAT1 sensitized AML cell to chemotherapy treatment. To study the role of NMNAT1 in leukemogenesis in vivo, we genetically deleted NMNAT1 in murine or human leukemia cells, transplanted them into recipient mice, and found that deletion of NMNAT1 reduced leukemic burden and extended leukemia-free survival (Fig. D).
Finally, to reveal the molecular mechanisms underlying NMNAT1 KO-mediated cell death (increased levels of gamma-H2AX), RNA-seq and functional assay of NAD+ dependent enzymes were performed. We found that the reduction of nuclear NAD+ resulting from NMNAT1 deletion upregulated genes involved in DNA repair pathway, which may be linked to impaired PARPs and Sirtuins activity. Our findings reveal the important function of NAD+ in leukemogenesis and chemoresistance, and identify NMANT1 as a novel therapeutic target for AML.
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Disclosures
No relevant conflicts of interest to declare.
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